Fig. 4: YY1 regulated PPP2CA expression through binding to PPP2CA promoter.
a YY1overexpression inhibited PPP2CA mRNA expression. CAL27 cells infected with GFP empty vector or PLVX-GFP-YY1 lentivirus and PPP2CA mRNA expressions were quantitated by real-time PCR. t-test, *P < 0.05 (n = 3). b Knockdown of YY1 upregulated PPP2CA mRNA expression. CAL27 cells stably transfected with scramble shRNA or YY1 shRNA and YY1 and PPP2CA mRNA expressions were quantitated by real-time PCR. t-test, *P < 0.05 (n = 3). c Knockdown of PPP2CA rescued YY1 knockdown-induced AKT T308 phosphorylation inhibition. Specific siRNA for PPP2CA was transfected into CAL27 cells that stably knockdown of YY1 for 48 h. Total proteins were extracted and subjected to Western blot analysis. The target bands were exposed, and densitometry was performed as fold change ratio from the mean of three independent experiments, one-way ANOVA: *P < 0.05 vs. control group. d YY1 overexpression inhibited PPP2CA promoter activity. PPP2CA promoter was transfected in CAL27 cells which stably infected with either PLVX-GFP-YY1 or GFP empty vector lentivirus for 24 h. e, f Knockdown of YY1 upregulated PPP2CA promoter activity. e CAL27 cells stably transfected with Tet-on YY1 shRNA and pretreated with doxycycline for 24 h and then transfected with PPP2CA promoter for another 24 h. f PPP2CA promoter was transfected into CAL27 cells which stably infected with either YY1 shRNA or scramble shRNA lentivirus for 24 h. d–f Total proteins were extracted and subjected to luciferase assay and normalized by total protein concentration. Data (mean ± SD) were presented as folds of control group. t-test, *P < 0.05 (n = 9). g, h YY1 bound to PPP2CA promoter in vivo. ChIP assays were performed in CAL27 cells with anti-YY1, anti-H3 or anti-IgG antibodies and with primers amplifying the −1464/−1303 and −1195/−994 region of the PPP2CA promoter containing YY1-binding sites A or B, respectively. ChIP samples was assessed by real-time PCR, data (mean ± SD) were presented as folds of anti-IgG group, one-way ANOVA: *P < 0.05 vs. Anti-IgG (n = 3). (I) YY1 bound to PPP2CA promoter in vitro. DAPA was performed using PPP2CA promoter −1373/−1358 and −1097/−1066 regions with YY1 binding site A and B respectively as DNA probes. The membrane was detected with YY1 antibody. j, k Deletion of YY1-binding site A almost abolished YY1 knockdown-induced upregulation of PPP2CA promoter activity. Deleted YY1-binding site was represented by crossed circle. Reporter constructs were transfected in CAL27 cells which stably transfected with Tet-on YY1 shRNA and treated with doxycycline or not for 24 h. Data (mean ± SD) were presented as folds of the non-treatment wild type group, one-way ANOVA: *P < 0.05 vs. non-treatment of wild type group; #P < 0.05 as indicated; N.S.: no statistical differences (n = 6)
